Rapid replacement of prevailing genotype of human respiratory syncytial virus by genotype ON1 in Beijing, 2012–2014

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Highlights

  • A PCR method was developed to identify the novel genotype ON1 of HRSV.

  • Rapid replacement of prevailing genotype by ON1 was found in Beijing, 2012–2014.

  • The evolutionary rate of the global ON1 strains was higher than previously estimated.

  • The time of most recent common ancestor of ON1 strains dated back to June 2009.

Abstract

Human respiratory syncytial virus (HRSV) is the most common viral pathogen causing lower respiratory infections in infants and young children worldwide. HRSV ON1 genotype in subgroup A with a characteristic of a 72 nucleotide duplication in the second highly variable region of attachment glycoprotein gene, has been reported in some countries since it was first detected in clinical samples collected in Canada in 2010. In this study, 557 HRSV antigen-positive nasopharyngeal aspirates were randomly selected during 2012/2013 to 2013/2014 HRSV seasons in Beijing for subgroup typing and for ON1 genotype screening by using a PCR based method developed for easily identifying genotype ON1 out of strains of subtype A. It was found that subgroup B was dominant in the 2012/2013 season and sudden shift of subgroup dominance from B to A and rapid replacement of previously prevailing NA1 genotype by ON1 genotype occurred in the 2013/2014 season. Reversible amino acid replacement in the G protein gene was found in a new branch of ON1 genotype. The evolutionary rate of the 351 global ON1 sequences was estimated to 7.34 × 10−3 nucleotide substitutions per site per year (95% highest probability density intervals, HPD, 5.71 × 10−3 to 9.04 × 10−3), with the time of most recent common ancestor dating back to June 2009.

Introduction

Human respiratory syncytial virus (HRSV) is the leading viral cause of severe pediatric respiratory tract infections worldwide, causing 1.9 million deaths per year (Williams et al., 2002). Repeated infections with HRSV are common throughout life (Henderson et al., 1979). The virus is composed of a non-segmented, single-stranded negative sense RNA genome packaged in a lipid envelope. It belongs to the genus Pneumovirus in family Paramyxoviridae of order Mononegavirales. Two major subgroups, HRSV-A and -B, have been identified by both antigenic and genetic analyses. The genome of HRSV is approximately 15.2 kb and contains 10 genes encoding at least 11 proteins (Collins and Crowe, 2007).

The attachment glycoprotein (G), one of the virus encoded transmembrane surface protein, is a type II integral protein, containing cytoplasmic domain, transmembrane domain and ectodomain (Roberts et al., 1994). The ectodomain is comprised of two mucin-like highly variable regions (HVR1 and HVR2) separated by a putative receptor-binding domain that is highly conserved within and between the two HRSV subgroups (Johnson et al., 1987). The G protein is involved in virus binding to cell membranes and is capable of stimulating neutralizing antibodies (Melero et al., 1997). Being the most variable protein, the G protein varies in its amino acid composition, length and distribution pattern of potential N- and O-linked glycosylation sites. These characteristics of the G protein may give HRSV the ability to evading the preexisted host immune response, facilitating recurrent infections and even large outbreaks. Thus, most of the studies on HRSV variability and evolution have been focused on G proteins, especially the HVR2.

By phylogenetic analyses for nucleotide sequences of HVR2, HRSV-A have been classified into 11 genotypes (GA1–GA5, GA6–GA7, SAA1, NA1–NA2 and ON1) and HRSV-B into 22 genotypes (GB1–GB4, BA1–BA6, BA7–BA10, BA-C, SAB1–SAB3, SAB4, URU1, URU2 and CB1) (Cui et al., 2013b). In 2003, Trento et al. (2003) reported three new HRSV-B strains isolated in 1999, named BA genotype, with the characteristic of a major change in the HVR2 introduced by a duplication of 60 nucleotides. Since then, BA genotype has gradually replaced other genotypes of HRSV-B and has become the predominant one worldwide (Trento et al., 2010). The duplicating segment was then used as a natural tag to track the global dissemination and evolution of HRSV (Trento et al., 2010). Coincidentally, a 72 nucleotide duplication in HVR2 nucleotide sequence was found in 10% of the subgroup A strains isolated in Ontario, Canada during 2010–2011 winter season (Eshaghi et al., 2012). HRSV strains with this 72 nucleotide duplication were designated genotype ON1. Up to date, HRSV genotype ON1 has been found in China (Cui et al., 2013b, Liu et al., 2014, Ren et al., 2014), Japan (Hirano et al., 2014, Tsukagoshi et al., 2013), South Korea (Lee et al., 2012), Germany (Prifert et al., 2013), South Africa (Valley-omar et al., 2013), Malaysia (Khor et al., 2013), Italy (Pierangeli et al., 2014), India (Choudhary et al., 2013), Thailand (Auksornkitti et al., 2013), Kenya (Agoti et al., 2014), Latvia (Balmaks et al., 2013), Spain (GenBank accession number: KF915266), Philippines (AB846656), Peru (KJ627264), Panama (KF301013), Croatia (KF057868) and USA (KJ672471).

The increasing frequency for detection of HRSV strains with ON1 genotype worldwide indicates that this genotype may have great advantages for escaping current host immunity. To easily screen and identify the strains with ON1 genotype, a PCR method was developed in this laboratory to find out the existence of this 72 nucleotide duplication. Rate of nucleotide substitution and the time of most recent common ancestor (TMRCA) of the global ON1 sequences were also estimated.

Section snippets

Ethics statement

This study was approved by the ethics committee of the Capital Institute of Pediatrics (CIP).

Sample collection

From July 2012 to May 2014, NPAs were collected and transported immediately to the Laboratory of Virology in CIP for the antigen detecting for respiratory viruses by using direct fluorescent assay (Diagnostic Hybrids, Athens, OH, USA). During this period, 1601 NPAs were HRSV antigen-positive, among which 612 (38.2%) were from girls and 989 (61.8%) from boys. Some of the samples were inoculated into

PCR for genotype ON1 screening

The PCR method developed in this study is able to distinguish strains of ON1 genotype from those of non-ON1 genotype in subgroup A as shown in Fig. 1A. Although the bands of PCR products from HRSV prototype strains A2 and Long appeared faint, the amplified products for non-ON1 (e.g. NA1) and ON1 genotype could be easily distinguished by different lengths of the products. By this method, 146 and 51 samples were identified as ON1 and non-ON1 genotypes, respectively, from 207 HRSV-A positive

Discussion

In this study, the rapid dissemination of ON1 genotype was found in Beijing, 2012–2014. The specific insertion of 72 nucleotide duplication in ON1 genotype was used as a tag to trace its global spread. The evolutionary rate and the TMRCA of ON1 genotype was estimated from global ON1 sequences to further illuminate the evolution of the virus.

The PCR method was developed to easily screen and identify the genotype ON1 from HRSV-A. It failed to identify ten out of the 207 strains. The reason of

Acknowledgments

We acknowledge the assistance of all the nurses and clinicians who performed sample collection. This work was supported by the Grant No. Z111107056811041 from the Beijing Municipal Science and Technology Commission and Grant No. 2013ZX10004-202 from Key Technologies R&D Program of National Ministry of Science.

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    Present address: Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, 1 Jianshedong Road, Zhengzhou, Henan 450002, China.

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